Environmental hazards, including UV irradiation and genotoxic chemicals, are some of the major sources for DNA damage and believed to be the major cause to human cancers and also a cause to many other diseases such as premature aging and neurodegeneration. DNA repair and DNA damage checkpoints are the two major biological defense systems against DNA damage in cells. Coordination between these two machineries is crucial for timely removal of DNA damage, preventing the conversion of DNA lesions to permanent mutations due to replication infidelity. However, the molecular details of the coordination remain largely unknown. As the major single-stranded DNA (ssDNA) binding protein in eukaryotes, replication protein A (RPA) is involved in almost all DNA metabolic pathways such as replication, recombination, DNA damage checkpoints, and all types of DNA repair pathways. Despite its critical activity in all these pathways the question of whether and how RPA plays a role in the precise coordination between DNA repair and checkpoints remain to be elucidated. Recent findings from our laboratory have shown that RPA undergoes hyperphosphorylatlon in response to DNA damage. In this project we will test the hypothesis that the hyperphosphorylatlon may alter the biochemical activity of RPA for interaction with ssDNA and proteins, and thus may constitute an important regulatory mechanism by which some DNA damage response interactions are inhibited while others are activated owing to the failure or success of the molecular interactions with RPA. To test these hypotheses, we will specifically determine: 1) the role of hyperphosphorylated RPA in the suppression of origin firing;2) the interaction between Rad51/Rad52 and RPA and its effect by RPA hyperphosphorylatlon;and 3) the cellular molecular interactions stimulated and/or disrupted by the hyperphosphorylation of RPA. The long-term goal of this project is to elucidate the molecular mechanism of DNA damage responses in human cells, and to better understand the damage-induced mutagenesis and related human diseases.